Non-Alcoholic Fatty Liver Disease (NAFLD) is a malady of increasing prevalence because of the growing population of individuals with obesity, metabolic syndrome and diabetes. This multifactorial disorder, due to both environmental and genetic factors, affects 30% of Americans with a prevalence of nearly 90% in obese individuals. NAFLD can lead to non-alcoholic steatohepatitis (NASH) with a prevalence of 25% in persons with obesity, and NASH in turn can lead to hepatocellular carcinoma, and cirrhosis. The pregnane X receptor (PXR) has emerged as a potential target for treatment of NAFLD as activation of this receptor results in hepatic steatosis in animal models. We have identified and characterized novel human pregnane X receptor antagonist FLB-12 that specifically disrupts the function of activated (agonist ligand-bound) PXR, but does not inhibit basal levels of PXR activity. This compound has shown to be a selective PXR antagonist in a variety of in vitro and in vivo models. More specifically and importantly FLB-12 was able to statistically improve an important disease component (hepatocyte ballooning) in a murine NAFLD model. However the potency and certain drug properties of FLB-12 need to be improved in order for this compound to be considered a viable predevelopment candidate. We will realize these goals by accomplishing the following specific aims: Aim 1: Identify structure-activity relationships (SAR) for allosteric antagonism of PXR to improve potency and ADME/PK properties. The goals of this aim are to increase PXR antagonist potency and selectivity as assessed by complementary in vitro assays already established in the Mani lab. Our objective is to reduce the IC50 values in each of these assays into the sub-micromolar range. Aim 2: Evaluate ADME/PK properties for PXR antagonists meeting criteria of Aim 1. We will explore potential drug properties by obtaining in vitro absorption, distribution, metabolism and excretion (ADME) data for up to 10 compounds. One or two of the most promising compounds will be evaluated for pharmacokinetic (PK) parameters in mice (IV administration) to determine in vivo terminal half-life, volume of distribution and clearance. These goals will be accomplished by combining the pharmaceutical and medicinal chemistry expertise of the scientists at the Fox Chase Chemical Diversity Center, Inc. (www.fc-cdci.com), the PXR structural biology expertise of Collaborations in Chemistry, and the extensive expertise of the Mani Lab at the Albert Einstein College of Medicine in the preclinical aspects of PXR modulators. Once we achieve the aims of this proposal, we will be well-positioned to transition into a lead optimization and full drug development program as part of the more extensive Phase II SBIR period of study where our goals would be to find preclinical drug candidates targeting PXR that we can evaluate in detailed NAFLD models under oral administration.